Human tissue plasminogen activator-human plasminogen activator inhibitor complex immunoassay and kit therefor

ABSTRACT

A kit for the immunological assay of the human tissue plasminogen activator-human plasminogen activator inhibitor complex in a human specimen, which kit comprises 
     (i) a monoclonal antibody (first antibody) against a human plasminogen activator inhibitor linked onto an insoluble solid carrier having a specular surface, 
     (ii) a polyclonal antibody (second antibody) against a human tissue plasminogen activator labelled by an enzyme, 
     (iii) a substrate and a reaction-discontinuing agent for the assay of the enzyme activity, 
     (iv) a diluent, and 
     (v) a detergent containing a nonionic surfactant having an HLB (Hydrophile Lipophile Balance) value of at least 16, 
     and a method for the immunological assay of the above complex and a method for the immunological assay of the active human plasminogen activator inhibitor, respectively in a human specimen using this kit.

Detailed Description of the Invention

a. Industrial Applicable Field

This invention relates to the immunological assay of the human tissueplasminogen activator-human plasminogen activator inhibitor complex in ahuman specimen. More detailedly, the invention relates to animmunological assay method for assaying with a high sensitivity andstably the amount of the complex in a human specimen, and a kittherefor.

The symbols used in the present description have the following meaningsunless otherwise defined.

tPA: Human tissue plasminogen activator

PAI: Human plasminogen activator inhibitor (Inhibitor against tPA)

tPA-PAI complex: Human tissue plasminogen activator-human plasminogenactivator inhibitor complex

b. Prior Art

Plasmin, an enzyme to dissolve fibrin, is formed from the conversion ofplasminogen with a tissue plasminogen activator (tPA). In recent years,it was found that an inhibitor against this human tissue plasminogenactivator [human plasminogen activator inhibitor (PAI)] exists in bloodvessel endothelial cells, cutaneous cells, platelets, the placenta,etc., and forms without delay a complex with the human tissueplasminogen activator, and thereby inhibits a human tissue plasminogenactivator activity (M. Philips et al, Biochem Biophys, Acta, 802,99-110, 1984, S. Thorsen, Biochem Biophys Acta, 802, 111-118, 1984, T.Wun et al, J, Biol Chem. 262, 3646-3653, 1987, M. A. Sanzo et al,Biochem, 26, 7443-7449, 1987, Y. Sakata et al, J. Biol Chem. 263,1960-1969, 1988). Further, there has been a clarification of therelationship between the activity value of the plasminogen activatorinhibitor (PAI) in the blood and a disease (B. Wiman et al. Scand. J.Clin Lab Invest 45, 43-43, 1985, P. Vague et al, Metabolism 35, 250-253,1986, A. Hamsten, Lancet 8549,3-8, 1987), and it is suggested that theplasminogen activator inhibitor (PAI) is an important control factor ofthe initiation mechanism of the blood coagulation fibrinogenolysissystem. Therefore, when it is possible to know the concentration of PAI,tPA and the tPA-PAI complex in the blood, there is a large possibilitythat the abnormality and pathema of the fibrinogenolysis system can bemonitored.

Presently, as assay methods for tPA there are methods by sandwich enzymeimmunoassay (Japanese Laid-Open Patent Publication No. 174759/1984),kits on the market (Biopool Co., IMULYSE t-PA), etc., and on the otherhand as methods for assay PAI there are methods using a radioactivesubstance (R. R. Schlef et al, J. Lab Clin Med 106, 408, 1985), asandwich enzyme immunoassay kit using a monoclonal antibody (BiopoolCo., IMULYSE PAI-I), etc.

Further, it is said that a very small amount of a tPA-PAI complex existsin the plasma, and as an assay method for this tPA-PAI complex there isan enzyme immunoassay method based on the fluorescence method, wherein amonoclonal antibody against PAI is used as an immobilized antibody, apolyclonal antibody against tPA is used as a labeled antibody and itsfragment is labeled with β-galactosidase.

This system to assay the tPA-PAI complex comprises reacting, first, theimmobilized monoclonal anti-PAI-1 antibody with a specimen at 30° C. for3 hours, washing, reacting with the Fab' fragment of the polyclonalanti-tPA labeled with β-galactosidase at 4° C. overnight to complete theimmunological logical reaction, decomposing 4-methyl-umbelliferylgalactoside, and assaying the formed 4-methylumbelliferone by thefluorescence method to quantitatively determine to tPA-PAI complex. Suchmethod has problems that the reaction temperatures differ between thetwo immunological reactions, reaction time is necessary to be overnightand very long, and further a fluorescence photometer, a specialmeasurement apparatus, is used, and thus it has been difficult to makethe assay effectively in industrial and clinical aspects.

On the other hand, Amiral et al. propose, as an assay method for atPA-PAI complex, a method for assaying the tPA-PAI complex using amonoclonal antibody against PAI and a monoclonal antibody against tPA(Thrombosis Research, Supplement VIII; 99-113, 1988). In this method twokinds of monoclonal antibodies are used and the tPA-PAI complex can beassayed with a relatively good sensitivity, but according to the ELISAdisclosed in this literature, the assay takes about 5 hours or more intotal. Therefore, in order to utilize actually and clinically the assaysystem wherein these two kinds of monoclonal antibodies are used, it isindispensable to further raise the assay sensitivity, simplify theoperation and shorten the operation time.

Problems to be Solved by the Invention

The first object of this invention lies in the provision of animmunological assay method and a kit, whereby the tPA-PAI complex in ahuman specimen can be assayed in a high sensitivity.

The second object of the invention lies in the provision of a method toassay the tPA-PAI complex in a human specimen by a convenient,clinically usable means.

Another object of the invention lies in the provision of animmunological method and a kit, whereby the tPA-PAI complex in a humanspecimen can be assayed stably and with a high sensitivity with minimalinfluence of subjects, assay conditions (time, temperature, etc.), etc.

Still another object of the invention lies in the provision of an assaymethod for the active PAI in a human specimen.

Still other objects of the invention will further be clarified by thefollowing description.

Means for Solving the Problems

According to studies by the present inventors, it was found that theabove objects and advantages of the invention can be accomplished by akit for the immunological assay of the tPA-PAI complex in a humanspecimen, which kit comprises

(i) a monoclonal antibody (first antibody) against a human plasminogenactivator inhibitor linked onto an insoluble solid carrier having aspecular surface,

(ii) a polyclonal antibody (second antibody) against a human tissueplasminogen activator labeled by an enzyme,

(iii) a substrate and a reaction-discontinuing agent for the assay ofthe enzyme activity,

(iv) a diluent, and

(v) a detergent containing a nonionic surfactant having an HLB(Hydrophile Lipophile Balance) value of at least 16.

Further, according to studies by the inventors are provided thefollowing immunological assay method for a tPA-PAI and assay method foran active PAI.

Immunological assay method for tPA-PAI (1) A method for theimmunological assay of the human tissue plasminogen activator-humanplasminogen activator inhibitor complex in a human specimen by

(1) contacting the human specimen with a first antibody linked to aninsoluble solid carrier, and then contacting a labeled second antibodytherewith, or

(2) simultaneously contacting a first antibody linked to an insolublesolid carrier, a labeled second antibody and the human specimen,

which method comprises

(a) using as the first antibody a monoclonal antibody against a humanplasminogen activator inhibitor linked onto an insoluble solid carrierhaving a specular surface,

(b) using as the second antibody a polyclonal antibody against a humantissue plasminogen activator labeled with an enzyme, and

(c) using as a detergent a detergent containing a nonionic surfactanthaving an HLB (Hydrophile Lipophile Balance) value of at least 16.

Assay method for an active PAI

In a method which comprises assaying respectively by an immunologicalassay method based on the sandwich method

(A) the human tissue plasminogen activator-human plasminogen inhibitorcomplex existing in a human specimen, and

(B) the human tissue plasminogen activator-human plasminogen inhibitorcomplex existing in the human specimen to which a human tissueplasminogen activator was added, and assaying the amount of the activehuman plasminogen activator inhibitor based on the difference of theassay values,

a method for the assay of the active human plasminogen activatorinhibitor which comprises

(a) using as a first antibody a monoclonal antibody against a humanplasminogen activator inhibitor linked onto insoluble solid carrierhaving a specular surface,

(b) using as a second antibody a polyclonal antibody against a humantissue plasminogen activator labeled with an enzyme, and

(c) using as a detergent a detergent containing nonionic surfactanthaving an HLB (Hydrophile Lipophile Balance) value of at least 16.

The above inventions are characterizing by

(i) using a monoclonal antibody against PAI as a first antibody(immobilized antibody) and an enzyme-labeled polyclonal antibody againsttPA as a second antibody in combination,

(ii) using as an insoluble solid carrier for the immobilization of thefirst antibody a solid carrier having a specular surface, namely anextremely smooth surface, and

(iii) using a detergent containing a nonionic surfactant having an HLBvalue of a certain value or more,

and the objects of the invention can be accomplished by the synergisticinfluences of these (i), (ii) and (iii). Namely, as the synergisticeffects of the above (i), (ii) and (iii), the nonspecific adsorption ofmany proteins existing in the human specimen, particularly tPA, PAI andthe like on the solid carrier is inhibited to the utmost, thenonspecific adsorption of the second antibody is inhibited and thespecific adsorption thereof is selectively promoted, and thus componentsunnecessary for the immune reaction can effectively be washed andremoved.

Thus according to the invention, tPA-PAI contained in an extremely smallamount in a human specimen can be assayed with a high sensitivity,conveniently, precisely and stably, and moreover, practical effects canalso be accomplished, i.e., the time required for the assay is sharplyshortened compared to usual methods and the use amount of the detergentis reduced.

Further according to the invention, when the immune reaction is carriedout by two steps, it is not necessary to set different temperatures ineach step and it is readily possible to carry out it at the sametemperature, and it is possible to carry out all steps including theimmune reaction and the enzymatic reaction in two hours or a shortertime.

Therefore, the immunological assay system in the invention, whereby ahuman specimen having abnormality in a coagulation fibrinogenolysisactivity or a human specimen suspected of having the abnormality caneffectively and speedily be assayed, has an extremely large clinicalsignificance.

This invention is described in more detail below.

[A] Preparation and isolation of an antigen

A monoclonal antibody against PAI and a polyclonal antibody against tPAused in the invention can be obtained using an antigen described below.

Namely, as tPA or PAI as an antigen for obtaining these antibodies therecan in principle be used either of one of a natural type extracted froma natural material and one of a recombinant type by a gene recombinanttechnique, but one obtained by a gene engineering method can also beused so long as it has immunological properties equal to the naturaltype tPA or PAI. Cell culture broths are used by choice as a materialfrom which a natural type tPA or PAI is obtained. Separation andpurification can be carried out by a combination of usually used proteinseparation techniques such as salting out, extraction, centrifugation,ultrafiltration and various chromatographies.

A polyclonal antibody or a monoclonal antibody can be prepared using asan antigen the thus obtained tPA or PAI.

[A]-(1) Preparation of a polyclonal antibody against tPA

A polyclonal antibody against tPA used in the invention can be obtainedaccording to a known method se using the tPA as an antigen. For example,as is described in "Edited by Nippon Seikagaku-kai (Japan BiochemistrySociety), Zoku Seikagaku Jikken Koza (Second Series BiochemicalExperiment Course), volume 5, pages 1 to 10, Tokyo Kagaku Dojin, 1986",an antiserum is obtained by immunizing an immunizable animal having anantibody production ability such as a guinea pig, rabbit, rat, mouse,goat with tPA by a conventional method and then withdrawing blood. Apurified antibody can be obtained from the antiserum by a combination ofusually used methods such as salting out, extraction, centrifugation,ultrafiltration and various chromatographies.

[A]-(2) Preparation of a monoclonal antibody

against PAI A monoclonal antibody against PAI used in the invention canbe obtained by using PAI as an antigen, culturing a hybridoma preparedby a cell fusion method by Kohler and Milstein known per se [G. Kohlerand Milstein, Nature (London), 256, 495-497 (1975)] to make it secretethe monoclonal antibody, and separating it from the culture broth.Namely, after the immunization of a mouse with PAI, the spleen cells ofthis mouse and a mouse myeloma cell are fused to prepare hybridomas.Since the thus obtained hybridomas produce various monoclonal antibodiesin accordance with the various fused lymphocytes, a hybridoma producinga desired monoclonal antibody is isolated as a hybridoma cloned bycloning. This cloned hybridoma is cultured in vitro or in the mouseabdominal cavity to make it secrete the monoclonal antibody. Themonoclonal antibody against PAI is separated from this culture brothaccording to conventional methods.

As such a monoclonal antibody against PAI, JTI-1, JTI-2, JTI-3 or JTI-4can be used as previously proposed by the present applicant anddisclosed in European Laid-Open Patent Publication No. 339302. Amongthem, JTI-3 and JTI-4 are preferred, and JTI-4 is particularly excellentin the invention.

These two monoclonal antibodies JTI-3 and JTI-4 are internationallydeposited according to the Budapest Treaty as described below.

A monoclonal antibody JTI-3 against PAI

As for this monoclonal antibody JTI-3 against PAI, the hybridomaproducing it was deposited with Fermentation Research Institute, aninternational deposit organ based on the Budapest Treaty, or Nov. 22,1988 as FERMp-10405, and thereafter changed to the international depositon Mar. 2, 1989 (International Deposit No. BP-2317).

This monoclonal antibody JTI-3 against PAI belongs to the subclass IgG₁and has a characteristic of recognizing to an antigenic determinationsite such that even when tPA was linked to PAI (namely even in a tPA-PAIcomplex), its linkage to PAI is not inhibited, but when the monoclonalantibody was linked to PAI, the linkage of tPA to PAI is inhibited.

A monoclonal antibody JTI-4 against PAI

As for this monoclonal antibody JTI-4 against PAI, the hybridomaproducing it is deposited with the above Fermentation ResearchInstitute, Agency of Industrial Science and Technology, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki-Ken 305, Japan, Nov. 22, 1988 asFERMp-10406, and thereafter changed to the international deposit(International Deposit No. Ferm BP-2318) on Mar. 2, 1989.

This monoclonal antibody JTI-4 against PAI belongs to the subclass IgG₁and its linkage to PAI is not inhibited even when PAI is linked to tPA.

[B] Preparation of an assay kit for a tPA-PAI complex [B]-(1)Preparation of a first antibody

As a first antibody in the invention, it is necessary to use amonoclonal antibody against PAI linked onto an insoluble solid carrierhaving a specular surface.

By using as this solid carrier one having a specular surface, namely onehaving an extremely smooth surface, nonspecific adsorption is loweredand the assay sensitivity of the tPA-PAI complex increases.

Heretofore, as insoluble solid carriers for highly sensitive assay,those whose surface area was enlarged by grinding and thereby rougheningthe surface have been used. Indeed, when the surface is enlarged, therearises a merit of increasing the amount of the immobilized antibody, butthere was a demerit that nonspecific adsorption is increased. As aresult of studies, the present inventors found based on the assay of atPA-PAI complex that a solid carrier having a specular surface, namelyhaving a center line average roughness (Ra) of 1.5 μm or less isadvantageous for the highly sensitive assay of the tPA-PAI complexbecause the amount of the antibody fixed thereon is almost equalcompared to a solid carrier having a roughened surface, whilenonspecific adsorption thereon is remarkably decreased. Although suchinsoluble solids having a specular surface are limited particularly inmaterial and shape, polystyrene beads and glass beads can for example bementioned.

The center line average roughness (Ra) means the value of Ra expressedby a micron unit which is given by the following equation when the partof the measurement length 1 is extracted from the roughness curve in thedirection of its center line, and the center line of this extractedpart, the direction of longitudinal magnification and the roughnesscurve are represented by an X axis, a Y axis and y=f(x) respectively,##EQU1##

About this center line average roughness (Ra) description is made JIS B0601-1982 (Japan), ANSI B 46.1-1979 (USA) and R468-1966 (ISOInternational Organization for Standardization).

In the following examples of the invention, the surface roughness of theinsoluble carriers was measured using a surface roughness testerSurfcom® produced by TOKYO SEIMITSU CO., LTD.

As monoclonal antibodies against PAI immobilized onto such inactivecarriers, there can be mentioned not only the above monoclonal antibodymolecules but also their fragments whose antigen likage ability is notlost, e.g. F(ab')₂, Fab, Fab', Facb (a half molecule of IgG), etc orderivatives of the antibodies or their fragments whose antigen linkageability is not lost. As methods to immobilize these antibodies ontoinsoluble carriers there can be used physical adsorption methods suchas, for example, a method which comprises immersing a polystyrenecarrier in a solution of such an antibody; ionic bond methods such as amethod using, for example, an ion exchange resin or a carrier having anionizing functional group such as an amine group, carboxylic acid group,sulfonic acid group or phosphoric acid group; covalent bond methods bychemical reactions such as, for example, carboxy-chloride methods,carbodiimide methods, maleic anhydride derivative methods, isocyanatederivative methods, cyanogen bromide-activated polysaccharide methods,diazo methods, active ester methods and carrier linkage methods usingcross-linking reagents (as crosslinking reagents there can be mentionedglutaraldehyde, hexamethylene isocyanate, a succinimide-maleimidecompound, etc.); and further methods wherein linkage is carried outthrough a substance having no linkage ability to PAI but capable ofbinding to the monoclonal antibody by a biological reaction, for examplea method using a protein A-bonded carrier, and the like.

[B]-(2) Preparation of a second antibody (labeled antibody)

In the invention, as a second antibody is used an enzyme-labeledpolyclonal antibody against tPA. Polyclonal antibodies against tPA ortheir fragments equal thereto described in the [A]-(1) are labeled usingenzymes used for immunological assay methods. As enzymes used for thislabeling, there can, for example, be mentioned lysozyme, maleatedehydrogenase, glucose-6-phosphate, dehydrogenase, peroxidaze, glucoseoxidase, alkaline phosphatase, luciferase, β-galactosidase, etc. Thelinkage between these enzymes and the polyclonal antibodies can becarried out by conventional methods such as glutaraldehyde methods,periodic acid methods and maleimide methods, but maleimide methods arepreferably used.

The maleimidation of antibodies and enzymes can be carried out bymethods known per se [Edited by Eiji Ishikawa "Koso Meneki Sokutei-ho"(Enzymatic Immunoassay Method), Igaku shoin], for example by using acrosslinking reagent such as succinimdyl4-(N-maleimidomethyl)cyclohexane carbonate (SMCC), sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane carbonate (sulfo SMCC), Succinimidylmetamaleimidobenzoate (MBS) or succinimidyl 6-maleimidohexanoate (EMCS).

Reaction between a polyclonal antibody against tPA or its fragment and alabeling substance can be carried out by reacting the polyclonalantibody maleimidated with an above crosslinking reagent or its fragmentwith an enzyme to which SH groups were introduced, for example withδ-acetylmercaptosuccinic anhydride, for example at a temperature of 4°to 30° C. and at a molar ratio of the antibody or its fragment to the SHgroup-introduced enzyme of 1:1 to 1:80 for 1 to 48 hours. In this case,it is preferred to obtain a product wherein the antibody or its fragmentis labeled with the labeling substance, chiefly in the rate of onemolecule of the latter per one molecule of the former through the sulfuratoms of the former.

[B]-(3) Detergent

In an immunological assay method according to the sandwich method, onlyby a simple combination of an immobilized antibody (first antibody) anda labeled antibody (second antibody), nonspecific adsorption takes placeand a highly sensitive assay system cannot be established. Thus, it is apoint for highly sensitive assay to inhibit the nonspecific reaction.For example, although the use of a surfactant in the assay systemreduces nonspecific adsorption, not all the surfactants are effective inthe tPA-PAI complex assay system.

The present inventors found that when the first antibody and the secondantibody are combined as described above, there arises an effect that aparticular surfactant does not inhibit the immune reaction in the assaysystem of the tPA-PAI complex but inhibits only the nonspecificadsorption of substances not involved in the immune reaction and thelabeled antibody.

In this invention, it is advantageous for highly sensitive assay to makesuch a surfactant as an immune activator exist in an immune reactionsolution or in a detergent solution. When the immune reaction is atwo-step reaction, the surfactant can be made to exist in eitherreaction but is preferably made to exist in the second reaction.

Surfactants used in the invention are nonionic surfactants having an HLB(Hydrophilic Lipophilic Balance) value of 16 or more.

Surfactants having an HLB below 16 are not preferred because theyinhibit the immune reaction as well as nonspecific adsorption.

Nonionic surfactants having an HLB valve of 16 or more include, forexample those of the polyoxyalkylene alkyl aryl ether series, thepolyoxyalkylene alkyl ether series, the polyoxyalkylene polyhydricalcohol fatty acid ester series, polyoxyethylene polyoxypropylene polyolseries, etc., and examples thereof are TRITON™ X-305 (HLB 17.3), TRITON™X-405 (HLB 17.9), EMULGEN™ 950 (HLB 18.2), EMULGEN™ 985 (HLB 18.9),TWEEN™ 20 (HLB 16.7), PLURONIC™ F68 (HLB 29), TETRONIC™ 707 (HLB >20),etc.

These surfactants can be used alone, or two or more surfactants can beused together, too.

The concentration of the surfactant in the detergent solution or thelike is 1 w/w % or less, preferably 0.001% or more and 0.1 w/w % orless.

In the invention, even in a surfactant amount of 0.1 w.w % or less, anexcellent washing effect is exerted, foaming at the time of washing isreduced, and as a result problems are diminished that foaming isintense, and thereby the removal of the foam becomes complicated, or itsremoval becomes incomplete and conversely washing becomes incomplete,and thus such a surfactant amount is preferred.

As a solvent for the nonionic surfactant any solvent can be used so longas it has no bad influence on the assay, and examples thereof are water,physiological saline and buffers such as a phosphate buffer.

[B]-(4) Diluent

As a diluent used in the assay kit and assay method of the invention,any diluent can be used so long as it is usually used in immunologicalassays. Namely, such a diluent is one having no bad influence on theimmune reaction, and for example are chiefly used buffers having a pH inthe range of 6.0 to 8.0 such as phosphate buffers, tris hydrochloridebuffers and acetate buffers.

[B]-(5) Substrate and reaction-discontinuing agent for enzymaticactivity assay

As substrates and reaction-discontinuing agents used in the assay kitand assay method of the invention, there can be used those usually knownin immunological assays in accordance with the kind of enzymes as alabeling substance. As examples thereof, examples of substrates ofperoxidase are 2,2'-azino-di-[3-ethyl-benzothiazolinesulfonic acid]diammonium salt (ABTS), orthophenylenediamine (OPD),3,3',5,5'-tetramethylbenzidine (TMB), etc., and examples ofreaction-discontinuing agents therefor are H₂ SO₄, HCl, acetic acid,glycine buffers (pH 10.3), sodium fluoride solutions, etc.

Examples of substrates of alkaline phosphatase are 4-nitrophenylphosphate, 4-methylumbelliferyl phosphate, NADP, etc.

Examples of substrates of β-galactosidase are2-nitrophenyl-β-D-glactoside, 4-methylumbelliferyl-β-D-glactoside, etc.Examples of reaction-discontinuing agents therefor are 0.1M Na₂ CO₃,etc.

[B]-(6) Use of protein

It was found by the research of the present inventors that when, in theassay of the tPA-PAI complex, a protein having a molecular weight of16,000 to 50,000, preferably 20,000 to 46,000 and an isoelectric pointof 1.0 to 5.0, preferably 1.2 to 4.8 is made to exist in the immunereaction solution and the final concentration of this protein in theimmune reaction solution is adjusted to 0.02 to 0.9% by weight,nonspecific adsorption is still further inhibited and thus background isremarkably lowered and further high sensitivity is easily obtained.Examples of such substances are casein, β-casein, α-casein, pepsin,ovoglycoproteins, orosomucoid, etc. These proteins can also be used as amixture thereof. Such a mixture can contain, for example, as maincomponents 10 to 60%, preferably 20 to 50% by weight of such a proteinand 30 to 80%, preferably 40 to 60% by weight of a sugar (e.g. lactose),and in addition a fat (e.g. 0.5 to 2% by weight), an ash (e.g. 5 to 12%by weight), water (e.g. 2 to 8% by weight), etc. Skim milk is a typicalexample of such a mixture. Skim milk contains casein as a protein, andhas advantages that it has a better dispersibility in the immunereaction solution, a higher nonspecific adsorption inhibition effect perunit weight of protein and a better preservability (precipitate is slowto be formed) at 4° C., compared to the case where casein is used alone.The source of skim milk used in the invention does not come intoquestion, and what is only required is that it is defatted milk. It ispreferred for the attainment of similar high sensitivity to make skimmilk exist in the immune reaction solution and adjust its ultimateconcentration in the immune reaction solution to 0.002 to 0.8% byweight. In a concentration lower than 0.002% by weight, a sufficientinhibition effect cannot be obtained, while a concentration higher than0.8% by weight in case of skim milk or 0.9% by weight in case of anabove protein inhibits specific reactions and is thus not preferred.

The above protein can be used in the immune reaction solution in theconcentrations mentioned above, and can be contained either in thediluent or in the secondary antibody.

[B]-(7) Assay method

Any human specimen can be used as the human specimen in the assay methodof the invention so long as it is a usual clinical liquid samplecontaining a tPA-PAI complex such as, for example, blood in the form ofserum or plasma, synovial fluid, lymph fluid, pleural effusion, ascites,amniotic fluid, cellular tissue fluid, bone marrow fluid or urine.Preferred is blood in the form of serum or plasma.

In the assay method of the invention, the amount of the tPA-PAI complexin a human specimen can be assayed in a high sensitivity by using thefirst antibody and the second antibody in a combination, and either

(i) contacting the human specimen with the first antibody linked to aninsoluble solid carrier, and after washing contacting the labeled secondantibody therewith, or

(ii) making the first antibody linked to an insoluble solid carrier, thelabeled first antibody, the labeled second antibody and the humanspecimen exist in a system.

The method (i) is not influenced by tPA even when tPA exists in a humanspecimen in a high concentration, e.g. in a specimen from a patient towhom tPA was administered for treatment, and thus superior to the method(ii).

Although the immune reaction temperature condition in the assay is notlimited so long as it does not denature the proteins as constituents anddoes not greatly inhibit the immune reaction, the reaction is suitablycarried out in general under a temperature condition on the order of 50°C. or less, preferably about 4° to 45° C. taking a time on the order ofabout 5 minutes to 5 hours, preferably 30 minutes to 3 hours.

For example, when the reaction temperature is a temperature close tobody temperature such as 37° C., nonspecific adsorption reaction cangreatly be diminished, and it is also one of the characteristics of thepresent invention system to exert great power for the high sensitizationof the assay system.

[C]Assay method of active PAI

The assay method for a tPA-PAI complex by the invention enables theassay of the tPA-PAI complex in a human specimen in an extremely highsensitivity, and thus by the utilization of this method, it becomespossible to assay the amount of the active PAI in the human specimeneasily and in a high sensitivity.

As a method for assaying the concentration of an active PAI, there hashitherto been reported by Takada et al. a method utilizing the formationof a tPA-PAI complex by reaction with tPA and PAI [Thrombosis Researchvol. 55, pages 285 to 589 (1989)]. According to this method, thereaction of PAI in a specimen with tPA is carried out at 4° C. overnightand the amount of the formed tPA-PAI complex is then assayed.

This method of assaying the tPA-PAI complex is carried out by firstcarrying out reaction at 30° C. for 3 hours, washing, conductingreaction with a polyclonal antibody tPA.Fab' fragment labeled withβ-galactosidase at 4° C., completing the immune reaction, decomposing4-methyl-umbelliferylgalactoside, and assaying the formed4-methylumbelliferone for fluorescence to quantatively determine thetPA-PAI complex. Effective assay thereof in view of industrial andclinical points has been difficult according to such a method becausethe method utilizes different temperatures in the two immune reactions,time necessary for the assay is two days and very long, and further afluorescence photometer, a special measurement apparatus is used.

On the other hand according to the invention, the amount of the activePAI in a human specimen can be assayed conveniently and in a short timeby adding tPA into the human specimen to react the active PAI with tPA,measuring the amount of the formed tPA-PAI complex and subtracting theamount of the complex in the specimen to which tPA was not added fromthe above amount.

Effect of the Invention

Thus according to the method of this invention, the tPA-PAI complex in aspecimen containing a very small amount of the tPA-PAI complex such as aclinical sample can quantitatively be determined in a high sensitivityand a high accuracy and by a convenient operation.

Further likewise, the amount of the active PAI in a specimen can beassayed in a high sensitivity and a high accuracy, simply and in a shorttime.

Example

The invention is detailedly described below by examples. The % symbol inthe examples denotes weight %.

DESCRIPTION OF DRAWING

FIG. 1 denotes a calibration curve for the immunological assay of thetPA-PAI complex, and denotes a relationship between the concentration ofthe tPA-PAI complex and absorbance.

FIGS. 2a and b denotes a relationship between the washing time andabsorbance in the immunological assay of the tPA-PAI complex.

FIG. 3 denotes a relationship between the degree of the surfaceroughness of the insoluble solid carrier and nonspecific adsorption ratein the immunological assay of the tPA-PAI complex.

REFERENCE EXAMPLE

[Preparation of peroxidase-labeled anti-tPA monoclonal antibody Fab'fragment]

The Fab' fragment of a monoclonal antibody against human tissueplasminogen activator (JTA-1; refer to European Laid-Open PatentPublication No. 339302) was prepared according to the method of Nisonov,and linked to peroxidase by a conventional method to obtainperoxidase-labeled Fab' (Editted by Japan Biochemistry Society, Lectureson Biochemistry Experiments, second series, volume 5, pages 109 to 112,Tokyo Kagaku Dojin, 1986). Namely, 40 μg of pepsin was added to 2 ml ofa 1.0 mg/ml solution of the monoclonal antibody [0.01M phosphoric acid-0.15M NaCl pH 7.2 (PBS)], and after adjustment to pH 3.7 with a 1Mcitrate buffer (pH 3.5) the mixture was subjected to digestion anddecomposition at 37° C. for one hour. 1N NaOH was added dropwise to thereaction solution to adjust its pH to 8 whereby the reaction wasdiscontinued. This reaction solution was subjected to TSK Gel G-3000SWcolumn (Toso)-HPLC using a 5 mM EDTA-0.1M phosphate buffer (pH 6.0) asan eluent to separate the F(ab')₂ fragment having a molecular weight of100,000.

This was concentrated by an ultrafilter. 200 μl of 0.1M2-mercaptoethylamine was added thereto and the mixture was subjected toreduction at 37° C. for 2 hours. This reaction solution was concentratedby an ultrafilter and subjected to HPLC in the same manner as in F(ab')₂to separate Fab' having a molecular weight of 50,000 and obtain 1.0 mlof a 1.07 mg/ml Fab' fragment solution.

On the other hand, 6.0 mg of horseradish peroxidase (Toyobo I-C) wasdissolved in 0.9 ml of a 1M sodium phosphate buffer (pH 7.0), 60 μl (2.9mg/80 μl ) of a dimethylformamide solution ofN-succinimidyl-3-maleimido-methyl-cyclohexane carbonate (Pierce Co.,SMCC) was added dropwise thereto, and reaction was carried out at 30° C.for 1 hour. This reaction solution was added to a SEPHADEX™ G-25 columnand a 0.1M sodium phosphate buffer (pH 6.0) was eluted to separatemaleimidated peroxidase. 3.2 mg of the thus obtained maleimidatedperoxidase and 1.0 mg of the Fab' fragment were reacted at 25° C. for 24hours, and the reaction mixture was subjected to separation andpurification using TSK Gel G-3000 SW column-HPLC (eluent 0.01M PBS) toobtain 0.35 mg of a peroxidase-labeled mouse anti-tPA antibody Fab'fragment having a molecular weight of about 130,000. The linkage molarratio of the Fab' fragment to peroxidase was 1:2 determined based on theabsorbances of the obtained labeled antibody at 280 nm and 403 nm.

EXAMPLE 1

[Preparation of peroxidase-labeled anti-tPA polyclonal antibody]

250 μl of a dimethylformamide solution (10 mg/ml) ofN-(m-maleimidobenzoic acid)-N-succinimide ester (MBS, Pierce Co.) wasadded to 5.0 ml of a PBS solution (1 mg/ml) of goat anti-human tPAantibody (Biopool Co.), and reaction was carried out at a temperature of25° C. for 30 minutes. Thereafter, gel filtration was carried out usinga column packed with SEPHADEX G-25 and a 0.1M phosphate buffer (pH 6.0)to separate the maleimidated polyclonal antibody from the unreacted MBS.

On the other hand, 197 μl of a dimethylformamide solution (60 mg/ml) ofS-acetyl succinic anhydride was added to 5.4 ml of a solution (10 mg/ml)of horseradish peroxidase (HRP) in a 0.1M phosphate buffer (pH 6.5). Themixture was subjected to reaction at a temperature of 25° C. for 30minutes, stirred for 4 minutes after the addition 2.2 ml of a 0.1M Trisbuffer (pH 7.0), 4.3 ml of a 1M-hydroxylamine aqueous solution and 0.4ml of a 0.1M EDTA aqueous solution, and subjected to gel filtrationusing a column packed with SEPHADEX G-25 and a 0.1M phosphate buffer (pH6.0) to separate SH lated peroxidase.

The MBS lated antibody and the SH lated peroxidase (5.3 mg) were reactedat 25° C. for 20 hours, and the reaction mixture was subjected to gelfiltration using HPLC (TSK Gel G-3000 SW) to obtain 1.9 mg of aperoxidase-labeled anti-tPA polyclonal antibody.

[Preparation of solid antibody]

Polystyrene beads (Immunochemical Co., D-7) having a center line averageroughness (Ra) of 1.35 μm were immersed in a solution (20 μg/ml) of themouse anti-PAI monoclonal anti-PAI antibody (JTI-4) in a 0.1M phosphatecitrate buffer (pH 3.0), and the mixture was allowed to stand at 4° C.for 20 hours. The beads were washed with a PBS solution, allowed tostand in a 1% BSA-PBS solution at room temperature for 2 hours, andwashed again with a PBS solution to obtain mouse anti-PAIantibody-immobilized beads.

[Making of calibration curve]

A human tPA-PAI complex was diluted with a 0.01M phosphate-0.5M NaCl-2%BSA buffer (pH 7.2) containing 0.25% skim milk to prepare 25, 12.5,6.25, 3.125, 1.56, 0 ng/ml solutions. 0.4 ml portions of the solutionsof each concentration and the mouse anti-PAI antibody-immobilized beadswere placed in small test tubes, and incubation was carried out at 37°C. for 1 hour. The respective kinds of beads were washed twice with awashing solution containing 0.05% TWEEN 20 in PBS.

A solution containing 1 μg/ml the peroxidase-labeled tPA polyclonalantibody was prepared using the antibody and a PBS-0.05% TWEEN 20solution. 0.4 ml portions of the solution were placed in the above smalltest tubes, followed by incubation at 37° C. for 30 minutes. After threetimes washing with the washing solution, 0.4 ml portions of a 2.5 mM H₂O₂ -0.025% 3,3',5,5'-tetramethylbenzidine 0.1M phosphate citrate (pH4.0) solution, a substrate solution for peroxidase, were added, andcolor was developed at 37° C. for 30 minutes. The color development wasstopped with 1N sulfuric acid and absorbance at 450 nm was measured.

The calibration curve is shown in FIG. 1.

EXAMPLE 2

[Comparison between the peroxidase labeled anti-tPA monoclonal antibodyand the anti-tPA polyclonal antibody]

A human tPA-PAI complex was diluted with a 0.01M phosphate-0.5M NaCl-2%BSA buffer (pH 7.2) containing 0.25% skim milk to prepare 25, 12.5, 6.25and 0 ng/ml solutions. 0.4 ml portions of the solutions of eachconcentration and the mouse anti-PAI antibody-immobilized beads preparedin Example 1 [Preparation of immobilized antibody] were placed in smalltest tubes, followed by incubation at 37° C. for 1 hour. After twicewashing with a washing solution containing 0.05% TWEEN 20 in PBS,adjustment was made using a PBS-0.05% TWEEN 20 solution so that theconcentration of the peroxidase labeled anti-tPA polyclonal antibodyobtained in Example 1 or the peroxidase-labeled mouse anti-tPA antibodyFab' fragment becomes 1 μg/ml, 0.4 ml portions thereof were placed inthe small test tubes, followed by incubation at 37° C. for 30 minutes.After two times washing with the washing solution, 3 ml portions of thewashing solution were placed in the small test tubes, and after allowingthe mixtures to stand for 0, 15, 20 and 30 minutes the washing solutionwas suction removed by an aspirator. Then, 0.4 ml portions of aperoxidase substrate (the same as in Example 1) were added, color wasdeveloped for 30 minutes, color development was stopped with 1N sulfuricacid, and absorbance was measured at 450 nm. The relation between thestanding time after the addition of the washing solution and absorbanceis shown in FIGS. 2a and b. FIG. 2a shows that when theperoxidase-labeled anti-tPA polyclonal antibody was used, the decreaseof absorbance is small and the influence by the time required for thewashing operation was insignificant.

EXAMPLE 3

Several kinds of polystyrene beads having a diameter of 6.35 mm anddifferent surface roughness were treated in the same manner as in"Preparation of immobilized antibody" in Example 1 to obtain severalkinds of mouse anti-PAI antibody-immobilized beads. A solutioncontaining a human tPA-PAI complex by 0 and 25 mg/ml was prepared with a0.25% skim milk-0.01M phosphate-0.5M NaCl-2% BSA buffer. 0.4 ml portionsof the solutions of each concentration and the plural kinds of mouseanti-PAI antibody-immobilized beads having a different surface roughnesswere placed in small test tubes, followed by incubation at 37° C. for 1hour. Washing was made twice with a washing solution containing 0.05%TWEEN 20 in PBS. TWEEN™ is a trademark for a series of general-purposeemulsifiers and surface active agents. They are polyoxyethylenederivatives of fatty acid partial esters of sorbitol anhydrides. 0.4 mlportions of the solution of the peroxidase-labeled anti-tPA polyclonalantibody in PBS-0.05% TWEEN 20 (1 μg/ml) obtained in Example 1 wereplaced in the small test tubes, followed by incubation at 37° C. for 30minutes. After washing was made three times with the washing solution,0.4 ml portions of the substrate solution for peroxidase were added,color was developed at 37° C. for 30 minutes, color development wasstopped with 1N sulfuric acid, and absorbance was measured at 450 mm.Nonspecific adsorption rate [(absorbance at 0 ng/ml/absorbance at 25ng/ml)×100] was determined from the obtained absorbance. The surfaceroughness of the polystyrene beads was measured by a surface roughnesstester Surfcom® (TOKYO SEIMITSU CO., LTD.). When the non-specificadsorption rate and the center line average roughness (Ra) as a measureof surface roughness are taken as the longitudinal axis and horizontalaxis respectively, FIG. 3 is obtained, and it is seen from the figurethat the smaller the surface roughness is, the smaller the nonspecificadsorption becomes. When the nonspecific adsorption rate was 2.5% orless, the center line average roughness was 1.5 μm or less.

EXAMPLE 4

The absorbance values at tPA-PAI concentrations of 0 ng/ml (N) and 25ng/ml (S) and their ratio (S/N) are denoted when solutions of thevarious surfactants in Table 1 in physiological saline were used as thewashing solution in "Making of calibration curve" in Example 1. When theS/N ratio of 40 is supposed to be the allowable limit of nonspecificabsorbance, the use of the nonionic surfactants having an HLB of 16 ormore is effective for the inhibition of nonspecific adsorption, as isshown in Table 1. However, P-8P and TWEEN 40, nonionic surfactantshaving an HLB of 16 or less inhibited nonspecific adsorption butinhibited at the same time the specific reaction (S), and was noteffective for highly sensitive analysis.

                                      TABLE 1                                     __________________________________________________________________________    S/N ratio at the time of the use of various surfactants                                            Concen-                                                                            Absorbance                                                               tration                                                                            tPA.PAI                                                                              tPA.PAI                                      Number Surfactant HLB                                                                              (w/w) %                                                                            (0 ng/ml) (N)                                                                        (25 ng/ml) (S)                                                                        S/N ratio                            __________________________________________________________________________    1      TWEEN 20™                                                                             16.7                                                                             0.1% 0.022  1.015   46.1                                 2      TWEEN 20™                                                                             16.7                                                                             0.05%                                                                              0.022  1.185   53.9                                 3      TWEEN 20™                                                                             16.7                                                                             0.01%                                                                              0.025  1.226   49.0                                 4      PLURONIC F63™                                                                         29.0                                                                             0.05%                                                                              0.023  1.203   52.3                                 Comparative                                                                   Example                                                                       1      P-8P       14.0                                                                             0.05%                                                                              0.025  0.895   35.8                                 2      TWEEN 40   15.6                                                                             0.05%                                                                              0.027  0.957   35.4                                 3      No         -- 0    0.122  1.235   10.1                                        addition                                                               4      SDS        40 0.05%                                                                              0.022  0.135   6.1                                  __________________________________________________________________________

EXAMPLE 5

[Inhibition of nonspecific adsorption with the addition of skim milk]

(a) Effect of skim milk addition to the first reaction solution

A human tPA-PAI complex was diluted with 0.01M PB-0.5M NaCl-2% BSA (pH7.2) buffers containing 0, 0.01, 0.125, 0.25, 0.5, 0.8 and 1.0% skimmilk respectively to prepare 0 and 25 ng/ml solutions. 0.4 ml portionsof each solution and the mouse anti-PAI antibody-immobilized beads wereplaced in small test tubes, followed by incubation at 37° C. for 1 hour.Washing was then made twice with a washing solution containing 0.05%TWEEN 20 in PBS. A solution of 1 μg/ml the peroxidase-labeled anti-tPApolyclonal antibody in a BPS-0.05% TWEEN 20 solution was prepared, and0.4 ml portions thereof were placed in the small test tubes, followed byincubation at 37° C. for 30 minutes. Washing was made three times withthe washing solution, 0.4 ml portions of the substrate solution forperoxidase were added, color was developed at 37° C. for 30 minutescolor development was stopped with 1N sulfuric acid, and absorbance at450 nm was measured. The results are shown in Table 2a. It is seen thatwhen the S/N ratio between the nonspecific adsorption (N) and thespecific reaction (S) of 40.0 is supposed to be an allowable limit, aneffective skim milk concentration range is 0.01 to 0.8%.

(b) Effect of skim milk addition to the second reaction solution

A human tPA-PAI complex was diluted with a 0.01M-PB-0.5M NaCl-2% BSA (pH7.2) buffer containing 0.25% skim milk to prepare 0 and 25 ng/mlsolutions. 0.4 ml portions of each solution and the mouse anti-PAIantibody solid beads were placed in small test tubes, followed byincubation at 37° C. for 1 hour. Then, washing was made twice with awashing solution containing 0.05% TWEEN™ 20 in PBS. Solutions inPBS-0.05% TWEEN 20 containing the peroxidase-labeled anti-tPA polyclonalantibody at a concentration of 1 μg/ml and containing respectively 0,0.01, 0.25, 0.5, 0.8 and 1.0% skim milk were prepared, and 0.4 mlportions thereof were placed in the small test tubes, followed byincubation at 37° C. for 30 minutes. After washing three times with thewashing solution, 0.4 ml portions of the substrate for peroxidase wereadded, color was developed at 37° C. for 30 minutes, color developmentwas discontinued with 1N sulfuric acid, and absorbance at 450 nm wasmeasured. The results are shown in Table 2b. The S/N ratio based on thenon-specific adsorption (N) and the specific reaction (S) increases bythe addition of skim milk by 0.01 to 0.8% and skim milk is seen to beeffective.

                  TABLE 2a                                                        ______________________________________                                        Inhibition of nonspecific adsorption by skim milk addition                    Skim milk      Absorbance                                                             concentration                                                                            tPA.PAI    tPA.PAI                                         Number  (%)        0 ng/ml(N) 25 ng/ml(S)                                                                            S/N                                    ______________________________________                                        1       0          0.185      1.390    7.5                                    2       0.002      0.033      1.388    42.0                                   3       0.01       0.026      1.388    53.4                                   4       0.125      0.025      1.361    54.4                                   5       0.25       0.023      1.328    57.7                                   6       0.5        0.023      1.320    57.4                                   7       0.8        0.024      1.201    50.0                                   8       1.0        0.026      0.936    36.0                                   ______________________________________                                    

                  TABLE 2b                                                        ______________________________________                                        Inhibition of nonspecific adsorption by skim milk addition                    Skim milk      Absorbance                                                             concentration                                                                            tPA.PAI    tPA.PAI                                         Number  (%)        0 ng/ml(N) 25 ng/ml(S)                                                                            S/N                                    ______________________________________                                        1       0          0.023      1.328    57.7                                   2       0.002      0.023      1.320    59.4                                   3       0.01       0.022      1.315    59.8                                   4       0.125      0.021      1.261    66.0                                   5       0.25       0.021      1.245    59.3                                   6       0.5        0.021      1.240    29.0                                   7       0.8        0.021      1.050    50.0                                   8       1.0        0.024      0.953    39.7                                   ______________________________________                                    

EXAMPLE 6

[Assay of specimens from normal persons and patients]

Assay was carried out using 4-fold dilutions of plasmas from 5 normalpersons and 3 patients of thrombosis in Example 1 [Making of calibrationcurve], and the results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Assay results on the specimens from                                           normal persons and thrombosis patients                                               tPA.PAI complex concentration (ng/ml)                                  ______________________________________                                        Normal                                                                        person                                                                        1        4.0                                                                  2        7.2                                                                  3        8.9                                                                  4        7.2                                                                  5        7.0                                                                  Patient                                                                       1        38.5                                                                 2        50.0                                                                 3        19.5                                                                 ______________________________________                                    

It is seen that the tPA-PAI complex concentrations in the thrombosispatients clearly exhibit high values and tPA-PAI complex concentrationcan be a marker in the prophylaxis of thrombosis.

EXAMPLE 7

[Assay of active PAI concentration]

A human tPA solution (0.5 μg/ml, 50 μl) was added to plasmas (each 50μl) from normal persons and thrombosis patients, followed by incubationat 37° C. for 30 minutes. Thereafter, the concentration of the tPA-PAIcomplex was determined (result A) according to the making of acalibration curve in Example 1. At the same time, there were determined(result B) the concentrations of the human tPA-PAI complex in theplasmas from the normal persons and the thrombosis patients to which tPAwas not added according to the making of calibration curve in Example 1.The concentration of the active PAI was determined by calculating thedifference of both assayed values and making molecular weight conversionof PAI (result C). The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                                (unit: ng/ml)                                                                   Active PAI                                          tPA.PAI complex concentration                                                                           Result C                                                   Result A     Result B      concent-                                    Specimen                                                                             (tPA addition)                                                                             (PA no addition)                                                                            ration*                                     ______________________________________                                        Normal                                                                        person                                                                        1      11.4         4.0           3.1                                         2      23.4         7.2           6.8                                         3      8.0          4.2           1.6                                         Throm-                                                                        bosis                                                                         patient                                                                       1      69.0         38.5          12.8                                        2      101.0        50.1          21.4                                        3      98.0         19.5          33.0                                        ______________________________________                                         *Active PAI concentration (Result A - Result B) × 0.42**                ##STR1##                                                                 

The active PAI concentrations in the thrombosis patients aresignificantly higher than those in the normal persons, and it wasdemonstrated that active PAI concentration can be a prophylactic markeron thrombosis.

We claim:
 1. A method for an immunological assay of a human tissueplasminogen activator-human plasminogen activator inhibitor complex in ahuman specimen by:(1) contacting the human specimen with a firstantibody linked to an insoluble solid carrier, and then contacting alabeled second antibody therewith, or (2) simultaneously contacting afirst antibody linked to an insoluble solid carrier and a labeled secondantibody with the human specimen, steps (1) and (2) being performed inthe presence of a surfactant, (3) and, following step (1) or (2),measuring said labeled second antibody bound to said solid carrier todetermine the human tissue plasminogen activator-human plasminogenactivator inhibitor complex in the human specimen, which methodcomprises:(a) using as the first antibody a monoclonal antibody whichspecifically binds a human plasminogen activator inhibitor JTI-4produced by the hybridoma JTI-4 (FERM BP-2318) linked onto an insolublesolid carrier having a specular surface, (b) using as the secondantibody a polyclonal antibody which specifically binds a human tissueplasminogen activator labeled with an enzyme, and (c) said surfactantbeing a nonionic surfactant having an HLB (Hydrophilic-LipophilicBalance) value of at least 16 and a concentration of 0.001-0.1 w/w %,said nonionic surfactant being at least one member selected from thegroup consisting of polyoxyalkylene alkyl aryl ether series,polyoxyalkylene alkyl ether series, polyoxyalkylene polyhydric alcoholfatty acid ester series and polyoxyethylene polyoxypropylene polyolseries.
 2. The method for the immunological assay of claim 1 whichcomprises contacting the human specimen with the first antibody linkedto the insoluble solid carrier, and then contacting with the labeledsecond antibody.
 3. The method for the immunological assay of claim 1 or2 wherein a nonspecific adsorption inhibiting protein having a molecularweight of about 16,000 to about 50,000 and an isoelectric point of 1.0to 5.0 contained in the second antibody and/or a diluent is furthercontacted such that the final concentration of said protein in theimmune reaction solution is adjusted to 0.02-0.9% by weight.
 4. Themethod for the immunological assay of claim 1 or 2 wherein the insolublesolid carrier is a polystyrene bead having a specular surface.
 5. Themethod for the immunological assay of claim 3 wherein the protein iscasein.
 6. An immunological assay for active human plasminogen activatorinhibitor which comprises:(1) providing two aliquots of a human specimencontaining (i) a human tissue plasminogen activator-human plasminogenactivator inhibitor complex and (ii) active human plasminogen activatorinhibitor, (2) adding a sufficient amount of free human tissueplasminogen activator to one of the two aliquots to complex the activehuman plasminogen activator inhibitor, (3) contacting both the aliquotsindividually with a first antibody linked to an insoluble solid carrier,and then contacting a labeled second antibody therewith, or (4)simultaneously contacting the first antibody linked to the insolublesolid carrier and the labeled second antibody with the aliquotsindividually, steps (3) and (4) being performed in the presence of asurfactant, (5) measuring solid phase bound label to determine theamount of human plasminogen activator-human tissue plasminogen activatorinhibitor complex in each of the aliquots, and (6) determining theamount of active human plasminogen activator inhibitor based on thedifference between the amount of human tissue plasminogenactivator-human plasminogen activator inhibitor complex in the twoaliquots, and wherein(a) there is used as the first antibody amonoclonal antibody JTI-4 produced by the hybridoma JTI-4 (FERM BP-2318)which specifically binds a human plasminogen activator inhibitor linkedto an insoluble solid carrier having a specular surface, (b) there isused as the second antibody a polyclonal antibody which specificallybinds a human tissue plasminogen activator labeled with an enzyme, and(c) there is used as the surfactant a nonionic surfactant having an HLB(Hydrophilic-Lipophilic Balance) of at least 16 and a concentration of0.001-0.1 w/w %, said nonionic surfactant being at least one memberselected from the group consisting of polyoxyalkylene alkyl aryl etherseries, polyoxyalkylene alkyl ether series, polyoxyalkylene polyhydricalcohol fatty acid ester series and polyoxyethylene polyoxypropylenepolyol series.